The present invention relates to a method of modifying polyphenol oxidase (PPO) activity in fruit and vegetables and to DNA sequences for use therein.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed. If the amount of this enzyme could be decreased the susceptibility of the tissue to brown would be reduced.
PPO sequence information may be used to construct synthetic genes which genes may be transformed into plants to decrease expression of the normal PPO gene, thereby decreasing synthesis of the enzyme.
It will also be understood that in certain instances the browning reactions in plants are desirable, such as in the production of black tea, cocoa, coffee, black pepper, black olives, etc. In these instances it may be desirable to increase the level of PPO to produce desired levels of browning or changes in flavour compounds.
The role of PPO in normal plant growth and development is not understood at present. There are a number of instances where increased levels of this enzyme are correlated with increased resistance to plant pathogens. It follow that genetic manipulation of plants to increase the level of PPO activity may confer useful resistance against pathogens and pests.
The grapevine PPO gene codes for an additional 103 amino acids upstream of the N-terminus of the mature protein. This region has the properties of a chloroplast transit peptide and is most likely responsible for targeting of the protein to be imported into the chloroplast and processed to produce the mature PPO protein. Transformation of plants with this gene may therefore result in correct targeting and maturation of the grapevine PPO in other species and result in accumulation of active grapevine PPO enzyme in the plastids of these tissues.
The terms xe2x80x9cgene encoding PPOxe2x80x9d, xe2x80x9cgene coding for PPOxe2x80x9d or xe2x80x9cPPO genexe2x80x9d as used herein should be understood to refer to the PPO gene or a sequence substantially homologous therewith.
In a first aspect of the present invention, there is provided a DNA sequence including a gene coding for a polypeptide having plant polyphenol oxidase (PPO) activity or a fragment thereof.
The DNA sequence may include a pre-sequence of a plant PPO gene coding for a transit peptide.
The DNA sequence may be modified. The DNA sequence may include a sequence coding for antisense mRNA to a plant PPO gene, or a fragment thereof.
The DNA sequence may include a catalytic cleavage site.
Alternatively the presequence may be replaced by other targeting sequences to direct the polypeptide having plant PPO activity to other cellular compartments.
The DNA sequence may include a putative chloroplast transit sequence and a mature grape vine PPO protein, as illustrated in FIG. 1.
The DNA sequence may include a gene coding for a polypeptide having a broad bean leaf PPO activity, as illustrated in FIG. 2.
The DNA sequence may include a gene coding for a polypeptide having apple fruit PPO activity, as illustrated in FIG. 3.
The DNA sequence may include a gene coding for a polypeptide having potato tuber PPO activity, as illustrated in FIG. 4.
In a further aspect of the present invention there is provided a DNA sequence including a sequence coding for antisense mRNA to a plant PPO gene, or a fragment thereof.
In a further aspect of the present invention there is provided a method for preparing a recombinant DNA plasmid including a DNA sequence coding for a polypeptide having plant PPO activity or a fragment thereof, which method includes
providing
a DNA sequence including a gene coding for a polypeptide having PPO activity or a fragment thereof; and
a plasmid expression vector; and
reacting the DNA sequence and the plasmid expression vector to deploy the DNA sequence within the plasmid expression vector.
The DNA sequence coding for PPO may be formed from polyadenylated RNA, for example isolated from a plant sample. The plant may be selected from apples, potatoes, grapes and beans. Preferably the plant sample is isolated from sultana grape berries, broad bean leaves, apple peel or cortex or potato tubers.
In order to provide a DNA sequence coding for PPO, in a preferred aspect of the present invention the method for the preparation of a recombinant DNA plasmid may include the preliminary steps of
providing a source of a polypeptide having plant PPO activity;
isolating polyadenylated RNA coding for a polypeptide having plant PPO activity therefrom; and
treating the polyadenylated RNA to construct copy DNA (cDNA).
The isolation of the polyadenylated RNA may be conducted utilising an oligo-dT spun column.
The step of treating the polyadenylated RNA to construct cDNA according to this aspect of the present invention may include
treating the polyadenylated RNA with reverse transcriptase and an adapter primer to form first strand cDNA; and
amplifying the cDNA so formed using the polymerase chain reaction (PCR).
The step of reacting the polyadenylated RNA with reverse transcriptase may utilise an oligonucleotide adapter primer having the sequence
5xe2x80x2-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT(SEQ ID NO: 15)
The step of amplifying the cDNA may utilise an adapter primer having the sequence
5xe2x80x2-GACTCGAGTCGACATCG(SEQ ID NO: 16) and a 5xe2x80x2-end primer.
The 5xe2x80x2-end primer may have the sequence
5xe2x80x2-CCIATICAGGCICCIGATATIICIAAGTGTGG(SEQ ID NO: 17) when utilized for the amplification of grape cDNA.
The 5xe2x80x2-end primer may have the sequence
5xe2x80x2-GCGGATCCTT[CT]TA[CT]GA[CT]GA[GA]AA[CT]AA(SEQ ID NO: 18).
when utilized for the amplification of bean cDNA.
The 5xe2x80x2-end primer may have the sequence
5xe2x80x2-GCGAATTCGA[AG]GA[TC]ATGGGIAA[TC]TT[TC]TA)(SEQ ID NO: 19)
when utilised for the amplification of apple cDNA.
The 5xe2x80x2-end primer may have the sequences
GEN3: (5xe2x80x2-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G)(SEQ ID NO: 20)
GEN7: (5xe2x80x2-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG)(SEQ ID NO: 21)
when utilised for the amplification of potato cDNA.
Alternatively, the step of treating the polyadenylated RNA to construct cDNA according to this aspect of the present invention may include
treating the polyadenylated RNA with reverse transcriptase and a PPO specific primer to form first strand cDNA;
treating the cDNA so formed with terminal d Transferase to attach a polyadenosine tail sequence at the 3xe2x80x2 end of the cDNA; and
amplifying the polyadenylated cDNA so formed by PCR.
The step of treating the polyadenylated RNA with reverse transcriptase may utilise a PPO specific oligonucleotide primer having the sequence
5xe2x80x2-AATCTTTGTGGTGACTGGCG(SEQ ID NO: 22)
for grape PPO or the PPO-specific primer is an oligonucleotide primer having the sequence
5xe2x80x2-GACGGTACATTAGTCTTAAAT(SEQ ID NO: 23)
for potato tuber PPO.
The step of amplifying the cDNA may utilise an oligonucleotide adapter primer having the sequence
5xe2x80x2-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT(SEQ ID NO: 15)
and a PPO specific oligonucleotide primer having the sequence
5xe2x80x2-ACCATCAGGCACGGTGGCGG(SEQ ID NO: 24)
for grape PPO or the sequence
5xe2x80x2-TGCTCATCAACTGGAGTTGAG(SEQ ID NO: 25)
for potato tuber PPO.
The plasmid expression vector for the cloning of th double stranded cDNA may be of any suitable type. The plasmid vector Bluescript SK+ has been found to be suitable.
The cloning step may take any suitable form. A preferred form may include
blunt-ending the cDNA, for example with Klenow fragment;
fractionating the cDNA so formed, for example on an Agarose gel;
isolating a fragment of the expected size, for example from the gel; and
ligating said fragment into a suitable restriction enzyme site, for example the HindIII or EcoRI site of a Bluescript SK+ vector.
In order to test the clones so formed, a suitable microorganism may be transformed with the plasmid expression vector, the microorganism cultured and the polypeptide encoded therein expressed. The microorganism Escherichia coli DH5 has been found to be suitable.
In a further aspect of the present invention there is provided a recombinant DNA plasmid including a DNA sequence coding for a polypeptide having plant PPO activity, or a fragment thereof, which plasmid is capable of being replicated, transcribed and translated in a unicellular organism.
The plasmid expression vector may be of any suitable type. The recombinant plasmid may contain a constitutive promoter element upstream of the DNA sequence coding for a polypeptide having PPO activity.
In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes
providing
a DNA construct including a modified gene coding for a polypeptide having plant PPO activity or fragment thereof; and
a plant sample; and
introducing said DNA construct into said plant sample to produce a transgenic plant.
The DNA construct may include a sequence encoding antisense mRNA to a plant PPO gene or a fragment thereof. The DNA construct may include a gene coding for a polypeptide having plant PPO activity or fragment thereof incorporating a catalytic cleavage site. X
The plant may be of any suitable type. In a preferred aspect the plant may be selected from the group including grapevine, potato, apple and bean.
In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes
providing
a DNA construct including a gene coding for a polypeptide having plant PPO activity or a fragment thereof; and
a plant sample; and
introducing said DNA construct into said plant sample to produce a transgenic plant.
The DNA construct may include a DNA sequence encoding a pre-sequence of a plant PPO gene or a fragment thereof.
The plant may be of any suitable type. In a preferred aspect the plant may be selected from the group comprising tobacco, broad bean, tomato, tea, coffee and cocoa.
The pre-sequence coding for the transit peptide may be replaced with other targeting sequences to direct the PPO protein to other cellular compartments. Sequences are already known which direct foreign genes into the vacuole, mitochondrion or intercellular space of plant cells. In addition the transit sequence for grapevine PPO could be used to target other proteins into the plastids.
The DNA construct may include a constitutive promoter which would result in expression of the introduced genes throughout the plant.
It will be understood that in many plant tissues PPO is highly expressed in certain tissue types. For example, PPO activity is much higher in the skin of grape berries than in the pulp, and the peel of potato tubers has higher activity than the cortex.
It may be desirable to alter levels of PPO activity only in certain plant tissues or at certain stages of plant development and this may be achieved by the use of specific promoter elements. For example, use of the patatin promoter alters PPO levels only in the tuber tissue of potato plants. This decreases PPO activity in the tuber, and reduce browning, but PPO activity in other parts of the potato plant is not altered.
Accordingly, the DNA construct may include a promoter which is specific to the peel or skin of fruit and vegetables to target foreign proteins specifically to the outer tissue layers.
This may allow properties of the skin or peel, such as colour, flavour, resistance to pathogens, etc to be manipulated independently of the inner parts of the fruit or vegetable which are consumed.
In a preferred aspect, the DNA construct may include a binary vector into which has been introduced a DNA sequence encoding PPO or a fragment thereof.
In a further preferred aspect, the introduction of the DNA construct into the plant may be by infection of the plant with an Agrobacterium containing the DNA construct.
In a further aspect of the present invention there is provided a transgenic plant, which plant contains a synthetic gene capable of modifying expression of the normal PPO gene.
The plant may be of any suitable type. In a preferred aspect the plant may be selected from the group comprising grapevine, potato, apple, tobacco, bean, peach, pear and apricot.
In a still further aspect of the present invention there is provided a plant vaccine including a sequence encoding PPO or a fragment thereof.
In a still further aspect of the present invention there is provided a DNA probe including a DNA sequence coding for a polypeptide having plant PPO activity or a fragment thereof.
The probe may be labelled in any suitable manner. A radioactive or non-radioactive labelling may be used. For convenience, the probe may be provided in the form of a cloned insert in a suitable plasmid vector.
The grapevine PPO sequence may be used to design general purpose oligonucleotide primers for use in the PCR to obtain this gene from other species. Plant PPO proteins are known to contain copper as a prosthetic group and two regions of the grape protein sequence which show homology to sequences from hemocyanin and tyrosinase proteins, corresponding to the copper binding sites on these proteins have been identified. Since these regions are apparently conserved between widely diverse organisms they are suitable for design of probes and primers to obtain other plant PPO genes.
Accordingly, in a still further aspect of the present invention there is provided a method of isolating a DNA sequence including a gene coding for a polypeptide having PPO activity or a fragment thereof from a plant species, which method includes
providing
a cDNA or genomic library; and
a DNA probe including a DNA sequence coding for a polypeptide having plant PPO activity or a fragment thereof; and
hybridising the probe with the genomic library to identify clones containing said DNA sequence.
The DNA probe may include a DNA sequence including, a fragment of the apple, potato, grape or bean PPO gene which is highly-conserved between different species.
The DNA probe may be prepared by a method which includes
providing
total cDNA from a plant species; and
two or more oligonucleotide primers which hybridise specifically with a gene coding for a polypeptide having plant PPO activity and which include sequences of the apple, potato, grape or bean PPO gene which are highly conserved between different species; and
performing PCT to amplify a DNA sequence including a gene coding for a polypeptide having plant PPO activity or a fragment thereof.
The oligonucleotide primers may include DNA sequences corresponding to the copper binding sites on the polypeptide having plant PPO activity.
In a still further aspect of the present invention there is provided a method of isolating a DNA sequence including a gene coding for a polypeptide having PPO activity, or a fragment thereof, from a plant species, which method includes
providing
mRNA isolated from the plant;
a poly-dT adapter primer; and
two or more oligonucleotide primers;
treating the mRNA with reverse transcriptase and an adapter primer to form first strand cDNA; and
amplifyig the cDNA so formed using the oligonucleotide primers and the polymerase chain reaction.
In a preferred aspect the oligonucleotide primers may be based on the apple, potato, grape or bean PPO gene sequences.
In a still further aspect of the present invention there is provided a method of isolating a DNA sequence including a gene coding for a polypeptide having PPO activity or a fragment thereof from a plant species, which method includes
providing
an expression library; and
a polyclonal antibody which has been raised against a purified polypeptide having PPO activity; and
reacting the polyclonal antibody with the expression library to identify clones containing a DNA sequence including a gene coding for a polypeptide having PPO activity or fragments thereof.
In a still further aspect of the present invention there is provided a method for purification of the PPO protein, which method includes
providing
a plant sample;
a detergent; and
one or more chromatography columns;
extracting the plant sample with the detergent;
treating the extract so formed with ammonium sulphate; and
fractionating the extract so formed by passing it through the chromatography columns.
The plant sample may be of any suitable type. The plant sample may be grapevine berries. This tissue contains high levels of PPO and in the juice of mature grape berries most of the PPO activity is bound to the solids and can be separated from the juice by centrifugation and then solubilised with detergents. The plant sample may be bean leaves.
The detergent may be cationic. The detergent hexadecyltrimethylammonium bromide (CTAB) has been found to be suitable.
The chromatography columns may be sepharose based. Three chromatography columns may be used. Q-sepharose followed by phenyl-sepharose followed by hydroxylapatite has been found to be suitable.
In a further aspect of the present invention there substantially pure form, having the N-terminal amino acid sequence
APIQAPDISKCGTATVPDGVTP(SEQ ID NO: 26)